We have made substantial progress on each of the aims of this R37. We outline here continuing plans- consistent with the original aims that also incorporate our own progress, the progress made by our collaborators and colleagues and technical advances arising in the last 5 years. Our budget reflects these original aims with a modest increase requested to accommodate new technologies and opportunities. The aims from the last competitive application are bolded as headers on each section. I) Genetics of non-syndromic clefts (case collection, subphenotyping, TDT, linkage) Our case ascertainment will continue in lowa and the Philippines and now include the more sophisticated subphenotyping we have initiated in collaboration with the Co-I on this project, Dr Marazita. We were fortunate to secure funding for ultrasound and 3D facial imaging instruments as part of a supplement to our now expiring P50 grant but there are no funds to subphenotype beyond this year, We will do the 3D imaging, 00 ultrasounds, dental and lip exams on the lowa and Philippines populations that have been solely supported by this grant. This enhanced phenotyping will benefit from the GWAS data retumed as part of our participation in Terrl Beaty's GWAS study that includes samples from both lowa and the Philippines. Additional genotyping of the extended family members with subphenotyping will then better inform gene finding and phenotype correlations. The lowa population Is now being studied for both clinical and tissue changes in wound healing. We will look at the IRF6, BMP4, TFAP2A and FOXE1 genotypes for the role of genotype in predicting wound healing response, an especially ripe area to tie to translational components. For linkage we have a third highly positive signal (after IRF6 and F0XE1)cir\ 6p and this is the subject of this grants current fine mapping efforts. II) Gene identification (linkage, LD, microdeletions, rnutation detection) We have built a terrific genomics collaboration with John Manak, a new Asst Prof In Iowa, who recently worked at Nimblegen on array technology. He has designed an array that tiles each of 10 cleft candidate genes at 10bp resolution to look for very small microdeletions in 100 cleft cases. The goal is to find small deletions marking regulatory regions that will provide opportunities for functional studies. As noted in progress (Rahimov, 2008) .we also have strong evidence for a second common allele for clefting in IRF6 in Asian populations and are now in the mutation discovery process for that variant as we also are for the contributory common allele in FOXE1. We had also identified a previously unsuspected cleft gene, .ADH1C, in the CIDR Derimark/Norway project and are in mutation discovery for that allele. Finally We have great emphasis on identifying the specific variants that appear to be conferring an increased risk for both clefts and Breast Cancer risk from FGFR2. We have preiiminary support for SNPs in F6FR2 and are doing cleft subphenotyping (00 defects) in a breast cancer population as well as functional analysis for the FGFR2 variants. This connection of clefting to adult outcomes would have siubstantial translational potential. III) Gene by gene and gene by environment studies We have now had success with G x E studies and we are expanding these using the lowa, Danish and Norwegian eampies (the Danish and Norwegian were collected under grant support now ended so work on them is supported by this R37). With the completed CIDR candidate gene work described in progress we are now doing the G x G work on these samples and can replicate findings on more than 2500 samples from the Philippines and over 9,000 case samples in total. IV) Long term studies (expression and animal models) Some of this work is underway such as our ongoing gene expression/functional work on IRF6 and the FGFs. In other cases it awaits new gene discovery and we will continue to collaborate with Schutte, Lidral, Fitzpatrick, Dixon and others we have worked with for many years and who have far more mouse model expertise. Continued Ancillary benefits of the R37 This R37 is the parent for Dr Felix's F32 Fogarty Award where she is investigating folate and homocysteine levels in mothers of children with clefts in Brazil. This is a critical extension to gene/environment interactions and will tie into folate trials underway as well. We would ernphasize that we also have supported an average of 8 honors undergraduate, dental or medical students per year on cleft projects in the lab. We hope we have established an environment in which students can explore paths to careers in dental/craniofacial research.